scholarly journals Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

2004 ◽  
Vol 167 (3) ◽  
pp. 505-518 ◽  
Author(s):  
Atsuo T. Sasaki ◽  
Cheryl Chun ◽  
Kosuke Takeda ◽  
Richard A. Firtel
Keyword(s):  
Actin Polymerization ◽  
Cell Movement ◽  
Leading Edge ◽  
Ras Signaling ◽  
Phosphatidylinositol 3 Kinase ◽  
Ras Activation ◽  
Directional Movement ◽  
Ras Effectors ◽  
Chemotaxis Receptors ◽  
Intermediate Event

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P3, the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P3 asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal.

scholarly journals Involvement of the Cytoskeleton in Controlling Leading-Edge Function during Chemotaxis

2010 ◽  
Vol 21 (11) ◽  
pp. 1810-1824 ◽  
Author(s):  
Susan Lee ◽  
Zhouxin Shen ◽  
Douglas N. Robinson ◽  
Steven Briggs ◽  
Richard A. Firtel
Keyword(s):  
Signaling Pathways ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Leading Edge ◽  
Ras Proteins ◽  
Ras Activation ◽  
Directional Movement ◽  
Edge Function

In response to directional stimulation by a chemoattractant, cells rapidly activate a series of signaling pathways at the site closest to the chemoattractant source that leads to F-actin polymerization, pseudopod formation, and directional movement up the gradient. Ras proteins are major regulators of chemotaxis in Dictyostelium; they are activated at the leading edge, are required for chemoattractant-mediated activation of PI3K and TORC2, and are one of the most rapid responders, with activity peaking at ∼3 s after stimulation. We demonstrate that in myosin II (MyoII) null cells, Ras activation is highly extended and is not restricted to the site closest to the chemoattractant source. This causes elevated, extended, and spatially misregulated activation of PI3K and TORC2 and their effectors Akt/PKB and PKBR1, as well as elevated F-actin polymerization. We further demonstrate that disruption of specific IQGAP/cortexillin complexes, which also regulate cortical mechanics, causes extended activation of PI3K and Akt/PKB but not Ras activation. Our findings suggest that MyoII and IQGAP/cortexillin play key roles in spatially and temporally regulating leading-edge activity and, through this, the ability of cells to restrict the site of pseudopod formation.

scholarly journals Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement

2007 ◽  
Vol 179 (7) ◽  
pp. 1539-1553 ◽  
Author(s):  
Rosa Ana Lacalle ◽  
Rosa M. Peregil ◽  
Juan Pablo Albar ◽  
Ernesto Merino ◽  
Carlos Martínez-A ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Cell Movement ◽  
Leading Edge ◽  
Cell Polarization ◽  
Type I ◽  
Lipid Kinase ◽  
Erm Proteins ◽  
Chemical Gradients ◽  
C Terminus ◽  
Phosphatidylinositol 4

Directional cell movement in response to external chemical gradients requires establishment of front–rear asymmetry, which distinguishes an up-gradient protrusive leading edge, where Rac-induced F-actin polymerization takes place, and a down-gradient retractile tail (uropod in leukocytes), where RhoA-mediated actomyosin contraction occurs. The signals that govern this spatial and functional asymmetry are not entirely understood. We show that the human type I phosphatidylinositol 4-phosphate 5-kinase isoform β (PIPKIβ) has a role in organizing signaling at the cell rear. We found that PIPKIβ polarized at the uropod of neutrophil-differentiated HL60 cells. PIPKIβ localization was independent of its lipid kinase activity, but required the 83 C-terminal amino acids, which are not homologous to other PIPKI isoforms. The PIPKIβ C terminus interacted with EBP50 (4.1-ezrin-radixin-moesin (ERM)-binding phosphoprotein 50), which enabled further interactions with ERM proteins and the Rho-GDP dissociation inhibitor (RhoGDI). Knockdown of PIPKIβ with siRNA inhibited cell polarization and impaired cell directionality during dHL60 chemotaxis, suggesting a role for PIPKIβ in these processes.

Osmotic stress activates Rac and Cdc42 in neutrophils: role in hypertonicity-induced actin polymerization

2002 ◽  
Vol 282 (2) ◽  
pp. C271-C279 ◽  
Author(s):  
Alison Lewis ◽  
Caterina Di Ciano ◽  
Ori D. Rotstein ◽  
András Kapus
Keyword(s):  
Actin Polymerization ◽  
Cell Movement ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Phosphatidylinositol 3 Kinase ◽  
Cytoskeleton Remodeling ◽  
Clostridium Difficile Toxin ◽  
Clostridium Difficile Toxin B ◽  
Gtpase Activation ◽  
Fold Activation

Hypertonicity inhibits a variety of neutrophil functions through poorly defined mechanisms. Our earlier studies suggest that osmotically induced actin polymerization and cytoskeleton remodeling is a key component in the hypertonic block of exocytosis and cell movement. To gain insight into the signaling mechanisms underlying the hyperosmotic F-actin response, we investigated whether hypertonicity stimulates Rac and Cdc42 and, if so, whether their activation contributes to the hypertonic rise in F-actin. Using a recently developed pull-down assay that specifically captures the active forms of these small GTPases, we found that hypertonicity caused an ∼2.5- and ∼7.2-fold activation of Rac and Cdc42, respectively. This response was rapid and sustained. Small GTPase activation was not mediated by the osmotic stimulation of Src kinases, heterotrimeric G proteins, or phosphatidylinositol 3-kinase. Interestingly, an increase in intracellular ionic strength was sufficient to activate Rac even in the absence of cell shrinkage. Inhibition of Rac and Cdc42 by Clostridium difficile toxin B substantially reduced but did not abolish the hypertonicity-induced F-actin response. Thus hypertonicity is a potent activator of Rac and Cdc42, and this effect seems to play an important but not exclusive role in the hyperosmolarity-triggered cytoskeleton remodeling.

scholarly journals Rear-polarized Wnt5a-receptor-actin-myosin-polarity (WRAMP) structures promote the speed and persistence of directional cell migration

2017 ◽  
Vol 28 (14) ◽  
pp. 1924-1936 ◽  
Author(s):  
Mary Katherine Connacher ◽  
Jian Wei Tay ◽  
Natalie G. Ahn
Keyword(s):  
Cell Migration ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Cell Movement ◽  
Leading Edge ◽  
Cellular Mechanism ◽  
Directional Movement ◽  
New Directions ◽  
And Function ◽  
Directional Cell Migration

In contrast to events at the cell leading edge, rear-polarized mechanisms that control directional cell migration are poorly defined. Previous work described a new intracellular complex, the Wnt5a-receptor-actomyosin polarity (WRAMP) structure, which coordinates the polarized localization of MCAM, actin, and myosin IIB in a Wnt5a-induced manner. However, the polarity and function for the WRAMP structure during cell movement were not determined. Here we characterize WRAMP structures during extended cell migration using live-cell imaging. The results demonstrate that cells undergoing prolonged migration show WRAMP structures stably polarized at the rear, where they are strongly associated with enhanced speed and persistence of directional movement. Strikingly, WRAMP structures form transiently, with cells displaying directional persistence during periods when they are present and cells changing directions randomly when they are absent. Cells appear to pause locomotion when WRAMP structures disassemble and then migrate in new directions after reassembly at a different location, which forms the new rear. We conclude that WRAMP structures represent a rear-directed cellular mechanism to control directional migration and that their ability to form dynamically within cells may control changes in direction during extended migration.

Myosin II-dependent cylindrical protrusions induced by quinine inDictyostelium: antagonizing effects of actin polymerization at the leading edge

2001 ◽  
Vol 114 (11) ◽  
pp. 2155-2165
Author(s):  
Kunito Yoshida ◽  
Kei Inouye
Keyword(s):  
Cell Membrane ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Cell Movement ◽  
Leading Edge ◽  
Contractile Vacuole ◽  
Cell Periphery ◽  
Slowing Down ◽  
On Line ◽  
Cylindrical Protrusion

We found that amoeboid cells of Dictyostelium are induced by a millimolar concentration of quinine to form a rapidly elongating, cylindrical protrusion, which often led to sustained locomotion of the cells. Formation of the protrusion was initiated by fusion of a contractile vacuole with the cell membrane. During protrusion extension, a patch of the contractile vacuole membrane stayed undiffused on the leading edge of the protrusion for over 30 seconds. Protrusion formation was not inhibited by high osmolarity of the external medium (at least up to 400 mosM). By contrast, mutant cells lacking myosin II (mhc− cells) failed to extend protrusions upon exposure to quinine. When GFP-myosin-expressing cells were exposed to quinine, GFP-myosin was accumulated in the cell periphery forming a layer under the cell membrane, but a newly formed protrusion was initially devoid of a GFP-myosin layer, which gradually formed and extended from the base of the protrusion. F-actin was absent in the leading front of the protrusion during the period of its rapid elongation, and the formation of a layer of F-actin in the front was closely correlated with its slowing-down or retraction. Periodical or continuous detachment of the F-actin layer from the apical membrane of the protrusion, accompanied by a transient increase in the elongation speed at the site of detachment, was observed in some of the protrusions. The detached F-actin layers, which formed a spiral layer of F-actin in the case of continuous detachment, moved in the opposite direction of protrusion elongation. In the presence of both cytochalasin A and quinine, the protrusions formed were not cylindrical but spherical, which swallowed up the entire cellular contents. The estimated bulk flux into the expanding spherical protrusions of such cells was four-times higher than the flux into the elongating cylindrical protrusions of the cells treated with quinine alone. These results indicate that the force responsible for the quinine-induced protrusion is mainly due to contraction of the cell body, which requires normal myosin II functions, while actin polymerization is important in restricting the direction of its expansion. We will discuss the possible significance of tail contraction in cell movement in the multicellular phase of Dictyostelium development, where cell locomotion similar to that induced by quinine is often observed without quinine treatment, and in protrusion elongation in general.Movies available on-line

scholarly journals WAVE3-mediated Cell Migration and Lamellipodia Formation Are Regulated Downstream of Phosphatidylinositol 3-Kinase

2005 ◽  
Vol 280 (23) ◽  
pp. 21748-21755 ◽  
Author(s):  
Khalid Sossey-Alaoui ◽  
Xiurong Li ◽  
Tamara A. Ranalli ◽  
John K. Cowell
Keyword(s):  
Cell Migration ◽  
Three Dimensional ◽  
Cell Movement ◽  
Leading Edge ◽  
Sh2 Domain ◽  
Regulatory Subunit ◽  
Phosphatidylinositol 3 Kinase ◽  
Cytoskeleton Reorganization ◽  
Lamellipodia Formation ◽  
Phosphatidylinositol 3

WAVE3 is a member of the WASP/WAVE family of protein effectors of actin reorganization and cell movement. The precise role of WAVE3 in cell migration and its regulation, however, have not been elucidated. Here we show that endogenous WAVE3 was found to be concentrated in the lamellipodia at the leading edge of migrating MDA-MB-231 cells. Platelet-derived growth factor (PDGF) treatment induced lamellipodia formation as well as two-dimensional migration of cells in the wound-closure assay and chemotactic migration toward PDGF in three-dimensional migration chambers. Knockdown of WAVE3 expression by RNA interference prevented the PDGF-induced lamellipodia formation and cell migration. Treatment of cells with LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), also abrogated the PDGF-induced lamellipodia formation and cell migration, suggesting that PI3K may be required for WAVE3 activity. WAVE3 and the PI3K regulatory subunit, p85, were found to interact in a yeast two-hybrid screen, which was confirmed through co-immunoprecipitation. The WAVE3-p85 interaction was mediated by the N-terminal region of WAVE3 and the C-terminal SH2 domain of p85. These results imply that the WAVE3-mediated migration in MDA-MB-231 cells via lamellipodia formation is activated downstream of PI3K and induced by PDGF. The findings of the WAVE3-p85 partnership also suggest a potential regulatory role for p85 in WAVE3-dependent actin-cytoskeleton reorganization and cell migration.

scholarly journals The Importance of Being PI3K in the RAS Signaling Network

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 1094
Author(s):  
Cristina Cuesta ◽  
Cristina Arévalo-Alameda ◽  
Esther Castellano
Keyword(s):  
Tumor Stroma ◽  
Ras Signaling ◽  
Ras Proteins ◽  
Cellular Processes ◽  
Oncogenic Ras ◽  
Ras Activation ◽  
Fda Approval ◽  
Effector Pathway ◽  
Ras Effectors ◽  
Tumor Types

Ras proteins are essential mediators of a multitude of cellular processes, and its deregulation is frequently associated with cancer appearance, progression, and metastasis. Ras-driven cancers are usually aggressive and difficult to treat. Although the recent Food and Drug Administration (FDA) approval of the first Ras G12C inhibitor is an important milestone, only a small percentage of patients will benefit from it. A better understanding of the context in which Ras operates in different tumor types and the outcomes mediated by each effector pathway may help to identify additional strategies and targets to treat Ras-driven tumors. Evidence emerging in recent years suggests that both oncogenic Ras signaling in tumor cells and non-oncogenic Ras signaling in stromal cells play an essential role in cancer. PI3K is one of the main Ras effectors, regulating important cellular processes such as cell viability or resistance to therapy or angiogenesis upon oncogenic Ras activation. In this review, we will summarize recent advances in the understanding of Ras-dependent activation of PI3K both in physiological conditions and cancer, with a focus on how this signaling pathway contributes to the formation of a tumor stroma that promotes tumor cell proliferation, migration, and spread.

scholarly journals GPCR-controlled membrane recruitment of negative regulator C2GAP1 locally inhibits Ras signaling for adaptation and long-range chemotaxis

2017 ◽  
Vol 114 (47) ◽  
pp. E10092-E10101 ◽  
Author(s):  
Xuehua Xu ◽  
Xi Wen ◽  
Douwe M. Veltman ◽  
Ineke Keizer-Gunnink ◽  
Henderikus Pots ◽  
...  
Keyword(s):  
Long Range ◽  
Molecular Mechanisms ◽  
Leading Edge ◽  
Negative Regulator ◽  
Ras Signaling ◽  
Gradient Sensing ◽  
Membrane Recruitment ◽  
Inhibitory Processes ◽  
Ras Activation ◽  
Wide Range

Eukaryotic cells chemotax in a wide range of chemoattractant concentration gradients, and thus need inhibitory processes that terminate cell responses to reach adaptation while maintaining sensitivity to higher-concentration stimuli. However, the molecular mechanisms underlying inhibitory processes are still poorly understood. Here, we reveal a locally controlled inhibitory process in a GPCR-mediated signaling network for chemotaxis inDictyostelium discoideum. We identified a negative regulator of Ras signaling, C2GAP1, which localizes at the leading edge of chemotaxing cells and is activated by and essential for GPCR-mediated Ras signaling. We show that both C2 and GAP domains are required for the membrane targeting of C2GAP1, and that GPCR-triggered Ras activation is necessary to recruit C2GAP1 from the cytosol and retains it on the membrane to locally inhibit Ras signaling. C2GAP1-deficientc2gapA−cells have altered Ras activation that results in impaired gradient sensing, excessive polymerization of F actin, and subsequent defective chemotaxis. Remarkably, these cellular defects ofc2gapA−cells are chemoattractant concentration dependent. Thus, we have uncovered an inhibitory mechanism required for adaptation and long-range chemotaxis.

Sign in / Sign up

Export Citation Format

Share Document